Acetylcholine (ACh), a neurotransmitter candidate in the central nervous system, is stored in multiple locations within a cholinergic nerve ending, the cytosol and the synaptic vesicles. The division of ACh into separate sites suggests that each site might have some functional significance. However, the respective physiological roles or the precise interrelationship between these pools requires further elucidation. Some investigators have suggested that the cytosol is the site of ACh formation and the vesicular fraction is the site of release. Other investigators have suggested that the cytosol is the site of both ACh formation and release. In both of these instances, the nerve ending is compartmentalized to provide one physiologically relevant form of ACh release. In contrast, the experiments described in this proposal will test the hypothesis that a central cholinergic nerve terminal is compartmentalized to provide multiple modes of transmitter release. In this model, ACh is not only stored in separate compartments but is also formed in and released from separate subcellular pools. Also, they will test whether the cytosolic pool of ACh is broken down to acetyl CoA and choline during depolarization of rat brain hippocampal tissue by reversal of the reaction catalyzed by the enzyme choline O- acetyltransferase (ChAT) to provide these substrates for the formation of that ACh released from the vesicular pool. Also, the experimental designs will test whether a soluble, cytosolic fraction of ChAT forms cytosolic ACh and whether a membrane associated fraction of ChAT, which requires detergent for solubilization, forms vesicular ACh. The experimental designs will test whether depolarization activates the detergent soluble fraction of ChAT associated with the vesicular fraction and whether such an activation is essential in maintaining the depolarized release of ACh. The significance of the proposed research will be to determine why ACh is stored, synthesized and released from separate sites within the central cholinergic nerve ending; also the potential interrelationship between these separate storage, synthetic and release sites.